eNOS-actin Interaction and Oxygen in Lung Endothelium

肺内皮细胞中的 eNOS-肌动蛋白相互作用和氧

• 批准号: 7882521
• 负责人: YUNCHAO SU
• 金额: $ 36.75万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882521
• 关键词: Actins; Adenoviruses; Arginine; Binding; Binding Sites; Biology; Blood Vessels; Cell Proliferation; Chronic Obstructive Airway Disease; Cytochrome c Reductase; Cytoskeleton; Data; Electron Transport; Endothelial Cells; Endothelium; Enzyme Kinetics; Gene Transfer; Hyperoxia; Hypoxia; Lead; Lung; Lung diseases; Mediating; Microinjections; Nitric Oxide; Oxidoreductase; Oxygen; Oxygen Therapy Care; Oxygen measurement, partial pressure, arterial; Patients; Peptides; Platelet aggregation; Play; Production; Proteins; Pulmonary Heart Disease; Pulmonary Hypertension; Reaction; Regulation; Reporting; Research; Role; Small Interfering RNA; Smooth Muscle Myocytes; Techniques; Technology; Testing; Vasodilation; angiogenesis; cytochrome c; ferricyanide reductase; human NOS3 protein; interdisciplinary approach; novel; protein aminoacid sequence; protein protein interaction; public health relevance; pulmonary artery endothelial cell; research study; yeast two hybrid system

DESCRIPTION (provided by applicant): Endothelial nitric oxide synthase (eNOS) is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms. We have found that eNOS is associated with the actin protein and that this association increases eNOS activity. We propose a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Aim #1: Determine whether actin interacts with the putative actin binding sequences (ABSs) in the eNOS protein resulting in increased electron transfer and catalytic activity of eNOS. We will use a yeast two-hybrid system and the peptides with sequences of the three putative ABSs of eNOS to pinpoint the actin-binding site in the eNOS protein. We will evaluate the effects of ABS peptides of eNOS on eNOS activity, eNOS-actin interaction, the enzyme kinetics, and on cytochrome c and ferricyanide reductase activities in the mixture of purified eNOS and actin to clarify the mechanism for increased eNOS activity by actin association. Aim #2: Determine whether alterations of eNOS activity in lung endothelium exposed to different oxygen tensions are due to changes in eNOS-actin association. We will manipulate the availability of actin in endothelium using siRNA and adenovirus gene transfer technology and introduce the ABS peptides of eNOS into endothelial cells to block eNOS-actin interaction using microinjection technique. Then we will observe hypoxia/hyperoxia-induced alterations in eNOS activity, the actin cytoskeleton, eNOS-actin association, NO release, and endothelium-dependent vasorelaxation. The successful completion of these aims will advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy. PUBLIC HEALTH RELEVANCE: This proposal is to study a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Proof of the role of eNOS-actin interaction in hypoxia/hyperoxia-induced alteration of eNOS function would advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy.

描述（由申请人提供）：内皮一氧化氮合酶（ENOS）受到各种转录，转录后和翻译后机制的严格调节。我们发现eNOS与肌动蛋白蛋白有关，并且这种关联增加了eNOS活性。我们提出了一个新的假设，即eNOS-肌动蛋白关联调节eNOS的电子转移，从而增加催化活性，并且eNOS-肌动蛋白相互作用的改变介导了暴露于不同氧气张力的肺内皮中eNOS的调节。 AIM＃1：确定肌动蛋白是否与eNOS蛋白中推定的肌动蛋白结合序列（ABS）相互作用，从而导致电子转移和eNOS的催化活性增加。我们将使用带有三个推定的eNOS腹部序列的酵母双杂交系统和肽来查明eNOS蛋白中的肌动蛋白结合位点。我们将评估eNOS的ABS肽对eNOS活性，eNOS-肌动蛋白相互作用，酶动力学以及细胞色素C和铁氰化物还原酶活性在纯化的eNOS和肌动蛋白的混合物中的影响，以阐明肌动蛋白关联的eNOS活性。目标＃2：确定暴露于不同氧气张力的肺内皮中eNOS活性的改变是由于eNOS-肌动蛋白缔合的变化所致。我们将使用siRNA和腺病毒基因转移技术来操纵肌动蛋白在内皮中的可用性，并将eNOS的ABS肽引入内皮细胞中，以使用微注射技术阻断eNOS-ACTIN相互作用。然后，我们将观察到缺氧/高氧诱导的eNOS活性改变，肌动蛋白细胞骨架，eNOS-肌动蛋白关联，无释放和内皮依赖性血管长期化。这些目标的成功完成将提高我们对NO生物学的理解，进而对以eNOS活性受损和血管NO产生的特征，例如原发性和继发性肺动脉高压，COPD和COR COR肺部具有重大影响。确定短期高氧的作用将有助于提高我们对氧疗法机制的理解，并为接受补充氧疗法的患者提供更好的管理策略。 公共卫生相关性：该建议是研究一个新的假设，即eNOS-肌动蛋白关联调节eNOS的电子转移，从而增加催化活性，并且eNOS-肌动蛋白相互作用的改变介导了暴露于不同氧气张力的肺内皮中eNOS的调节。 eNOS-肌动蛋白相互作用在缺氧/高氧诱导的eNOS功能变化中的作用证明将提高我们对NO的生物学的理解，进而对eNOS活性和血管活性受损的肺部疾病产生重大影响，例如原发性和二级肺部和肺部高血压，COPD，COPD和CORNEALE。确定短期高氧的作用将有助于提高我们对氧疗法机制的理解，并为接受补充氧疗法的患者提供更好的管理策略。